Carvacrol treatment opens Kir6.2 ATP-dependent potassium channels and prevents apoptosis on rat testis following ischemia–reperfusion injury model

Abstract

Testicular torsion is a urological problem that causes subfertility and testicular damage in males. Testis torsion and detorsion lead to ischemia–reperfusion (IR) injury in the testis. Testicular IR injury causes the increase of reactive oxygen species (ROS), oxidative stress (OS) and germ cell-specific apoptosis. In this study, we aimed to investigate whether Carvacrol has a protective effect on testicular IR injury and its effects on Kir6.2 channels, which is a member of adenosine triphosphate (ATP)-dependent potassium channels. In the study, 2–4 months old 36 albino Wistar rats were used. For experimental testicular IR model, the left testis was rotated counterclockwise at 720° for two hours, and after two hours following torsion, detorsion was performed. Carvacrol was dissolved in 5% Dimethyl Sulfoxide (DMSO) at a dose of 73 mg/kg and half an hour before detorsion, 0.2 mL was administered intraperitoneally. In testicular tissues, caspase 3 and Kir6.2 immunoexpressions were examined. Serum malondialdehyde (MDA) and testosterone levels were measured. Apoptotic cells and serum MDA levels were significantly decreased and Kir6.2 activation was significantly increased in Carvacrol-administrated IR group. As a result of our study, Carvacrol may activates Kir6.2 channels and inhibits apoptosis and may have a protective effect on testicular IR injury.

⧉ Introduction

Testicular torsion is a urological problem that causes testicular damage and subfertility by rotating around the testicular vessels and spermatic cord of the testicle [1]. Testicular torsion or spermatic cord torsion is one of the most serious urological emergencies and it is usually seen in newborns and young men. If testicular torsion is misdiagnosed or improperly treated, it leads to the development of male subfertility [2]. The testicle suddenly rotates in the scrotum, bending the blood vessels inward and blood flow is stopped. If intervention is not performed to testicular torsion more than 4–8 hours; it causes severe pain and can lead to testicular necrosis. Testicular torsion incidence is approximately 5/100 000 men under the age of 25 [3]. Testicular torsion in animal models, such as rat, represents a functional model of ischemia–reperfusion (IR) in humans [4]. In testicular torsion, there are two events that cause testicular necrosis. The first is ischemic damage during torsion, the second is reperfusion damage during detorsion. With the cessation of blood flow by testicular torsion, ischemia occurs, energy-rich phosphates are depleted and the level of products, such as hypoxanthine, increases [5,6]. Following testicular detorsion, reperfusion occurs in testicular tissue which causes more serious damage than ischemia [7]. Reperfusion damage occurs with the increase of free oxygen species and neutrophils [8].

IR injury causes cellular damage by the formation of free radical species, proinflammatory cytokines, adhesion molecules, lipid peroxidation, apoptosis, anoxia, and alteration of microvascular blood flow. This chain of pathological events causes atrophy in the testicle [8]. Oxidative stress (OS) associated with overproduction of reactive oxygen species (ROS) forms the basic pathophysiological process in IR injury [9,10]. ROS attack polyunsaturated fatty acids in the cell membrane, which causes a series of chain chemical reactions called lipid peroxidation [11]. Lipid peroxidation in the cell membrane damages the cells by disrupting the fluidity and permeability of the cell membrane [12]. In other words, when the cell membrane is damaged by free radicals, cell death may occur. Malondialdehyde (MDA) is released during lipid peroxidation and used as a marker of peroxidative damage; it can be detected via various biochemical assays [13,14,15]. MDA molecules penetrate cell membrane structures and leads to asymmetric disorders in lipid membrane components [12]. Lipid peroxidation damage on testicular dysfunction has been known for a long time [16]. This damage impairs mid part of sperm and reduces acrosome capacitation during fertilization [12]. Testicular IR causes germ cell apoptosis [17]. During IR high ROS levels induce caspase activation and apoptosis by disrupting the internal and external mitochondrial membranes and it causes the release of cytochrome c protein. There is a correlation between increased ROS levels and high levels of cytochrome c, caspase 9 and caspase 3 which indicate apoptosis in infertile male patients [18].

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels are hetero-octameric proteins consisting of KIR 6.x-type subunits and Sulfonylurea receptor (SUR) subunits [19]. It was shown that KATP channel subunits are found in the cell membrane, organelles of spermatogenic cells and Sertoli cells in mouse and rat testis [20,21]. Potassium channels are responsible for maintaining the membrane potential of germ cells. Different potassium channels are localized in germ cells at different stages [22]. ATP-dependent potassium channels are formed by two types of molecules, a pore-forming subunit molecule Kir6.x (Kir6.1 and Kir6.2) and a SUR regulatory subunit molecule [23,24]. It is shown that KATP channel subunits Kir6.1, Kir6.2 and SUR2B are found highly in round spermatids and SUR2A in long spermatids [20]. Kir6.2 is known to be localized in spermatids together with SUR2B. These channels may play a role in capacity-related sperm hyperpolarization [21]. Also, it has been determined that opening of the KATP channels can protect the tissue against IR damage [19]. Many testicular IR model studies showed that spermatogenesis activation was decreased. This loss in spermatogenesis has been defined as germ cell-specific apoptosis [17]. It has been reported that KATP channels were opened in testicular tissue during IR injury, and it prevented caspase-dependent apoptosis [25].

Carvacrol [2-methyl-5-(1-methylethyl)-phenol] is a monoterpenoid compound with a characteristic Oregano smell, found in essential oils of aromatic plants, such as Thyme, Pepper, and Wild Bergamot [26]. In vitro and in vivo studies described that Carvacrol has different bioactivities, such as antibacterial, antioxidant, antiseptic, antispasmodic, growth regulator, antifungal, antiviral, anti-inflammatory, expectorant, antitussive, immunomodulator and chemical inhibitor, rumen microbial fermentation modifier and reduction of Methane emission [27,28,29]. Studies have shown that antioxidants protect spermatozoa from ROS, preventing abnormal spermatozoa production. Antioxidants reduce ROS produced by leukocytes, prevent deoxyribonucleic acid (DNA) fragmentation, improve sperm quality, prevent premature sperm maturation [30]. Carvacrol plays a critical role as a natural antioxidant in reducing lipid peroxidation [31].

Aim

Based on the literature, we hypothesized that Carvacrol has a protective role against testicular IR damage. Therefore, we aimed to investigate the effects of Carvacrol administration on testicular IR injury morphologically, immunohistochemically, and biochemically.

⧉ Materials and Methods

Animals

Two–four months old 36 male Wistar albino rats, weighing between 200–250 g, were placed in a temperature (21±2°C) and humidity (60±5%) controlled room in which a 12-hour light–dark cycle was maintained. Animals were fed with standard laboratory food and tap water in type 3H cages. Experimental animals were procured from Experimental Animals Research Center, Bolu Abant Izzet Baysal University Bolu, Turkey. This study was approved by the Ethics Committee of Experimental Animal Studies, Bolu Abant Izzet Baysal University (Approval No. 2017/08) and were in accordance with the Declaration of Helsinki and International Association for the Study of Pain Guidelines. In this study, rats were randomly divided into six groups, each containing six rats. Experimental groups were determined as Sham, Dimethyl Sulfoxide (DMSO), Carvacrol, IR, IR+DMSO and IR+Carvacrol. Before experiment, animals were anesthetized with an intraperitoneal (i.p.) injection of 0.25 mL Ketamine hydrochloride (75 mg/kg) and 0.1 mL Xylazine (8 mg/kg).

Experimental design and testicular IR model

Testicular IR model in rats were performed in Experimental Animals Unit, Faculty of Medicine, Bolu Abant Izzet Baysal University. We used power analysis to determine sample size and included six rats in each experimental group. There was no animal death due to experimental procedure.

Sham group (n=6)

A Sham operation (rats that underwent only scrotal incision and suturing) was performed on rats. The scrotum of the rats was opened under anesthesia and the left testicle was taken out. Without any experiment, the testicle was put into the scrotum and the scrotum was sutured. After waiting for four hours based on IR time, left testicle tissue was taken.

DMSO group (n=6)

0.2 mL of 5% DMSO (Sigma D8418, 50 mL) was injected i.p. under anesthesia. Left testicular tissue was taken at the end of 2.5 hours after i.p. injection.

Carvacrol group (n=6)

73 mg/kg Carvacrol [32] (Sigma W224511-100G-K) dissolved in 5% DMSO was injected 0.2 mL i.p. Left testicular tissue was taken at the end of 2.5 hours.

IR group (n=6)

Torsion was created by rotating the left testis 720° counterclockwise and then two hours later for detorsion, the testis was counter-rotated to the natural position and replaced into the scrotum. Left testicle tissue was removed after two hours of reperfusion injury.

IR+DMSO group (n=6)

Torsion was created by rotating the left testis 720° counterclockwise for two hours ischemic injury. Half an hour before detorsion, 0.2 mL of 5% DMSO was injected i.p. Then two hours later from torsion beginning, the testis was counter-rotated to the natural position and replaced into the scrotum for detorsion. Left testicle was removed after two hours of reperfusion injury.

IR+Carvacrol group (n=6)

Torsion was created by rotating the left testis 720° counterclockwise for two hours ischemic injury. Half an hour before detorsion, 73 mg/kg Carvacrol was dissolved in 5% DMSO and injected i.p. Then two hours later from torsion beginning, the testis was counter-rotated to the natural position and replaced into the scrotum for detorsion. Left testicle was removed after two hours of reperfusion injury.

All testicular tissues were placed in 10% neutral buffered formalin. Left epididymis was taken into phosphate-buffered saline (PBS) solution for spermiogram. Approximately 3 mL of blood was taken from tail vein of each animal and placed in blood tubes. After obtaining tissue and blood samples, rats were sacrificed via application of high-dose anesthetics. Blood tubes were centrifuged at 9000 rpm for 10 minutes. The serum obtained were put into tubes and kept at -80°C for biochemical analysis.

Histological analysis

The collected tissue samples were fixed in 10% neutral buffered formalin for 24 hours. Following fixation, tissues washed with water for 30 minutes to remove fixation solution and tissues were dehydrated in solution containing an increasing percentage of ethanol. Following dehydration, tissues were passed through xylene to remove ethanol and clear tissues. After tissue processing, tissues were embedded in paraffin. Formalin-fixed paraffin-embedded (FFPE) tissues were cut into 5 μm sections and placed on slides. Sections were stained with Hematoxylin–Eosin (HE) and Masson’s trichrome for histological analysis. After stainings, testicular tissues of all groups were investigated for histopathological (HP) changes, such as interstitial edema, interstitial bleeding, and hemorrhagic infarction.

The seminiferous tubules were graded according to the Johnsen’s score [33]. Tubuli in 20 consecutive 200× field areas were scored, and mean values were determined. The seminiferous tubule diameters were measured in all groups. Johnsen’s score is based on the premise that with testicular damage there is successive disappearance of the most mature cell type; progressive degeneration of germinal epithelium; and with disappearance of sperm and spermatids, then spermatocytes, and finally Sertoli cells (in that order). A score of 1 indicates no seminiferous epithelial cells and tubular sclerosis. Score 2: no germ cells, only Sertoli cells. Score 3: spermatogonia only. Score 4: no spermatids, few spermatocytes and arrest of spermatogenesis at the primary spermatocyte stage. Score 5: no spermatids and many spermatocytes. Score 6: no late spermatids, few early spermatids, arrest of spermatogenesis at the spermatid stage, and disturbance of spermatid differentiation. Score 7: no late spermatids and many early spermatids. Score 8: few late spermatids. Score 9: many late spermatids and disorganized tubular epithelium. Score 10: full spermatogenesis.

Immunohistochemistry (IHC)

FFPE samples were cut into 5 μm sections and placed on slides coated with poly-L-Lysine. Sections were incubated with primary antibodies anti-caspase 3 (Thermo Scientific™ caspase 3 (CPP32) Ab-4, rabbit polyclonal antibody) (1:200) and anti-Kir6.2 (Santa Cruz Kir6.2 (B-9) sc-390104, mouse monoclonal antibody), overnight, at 4°C. The slides were incubated with Horseradish Peroxidase (HRP)-conjugated secondary antibody (Thermo Scientific™ Lab Vision™ UltraVision™ Detection System (TP-015-HD), anti-polyvalent antibody) for 40 minutes. Peroxidase activity was visualized with 3,3’-Diaminobenzidine (DAB) (Catalogue #D4168 – SIGMAFAST™ DAB tablets), and counterstained with Hematoxylin. Sections were evaluated and photographs were taken using a light microscope (Nikon Eclipse i80, Tokyo, Japan). The images were then analyzed using NIS-Elements Microscope Imaging Software (Nikon, Tokyo, Japan). The number of positively stained cells on seminiferous tubule was counted by a blinded manner by two independent observers in five random fields in the slides from each tissue samples.

Terminal deoxynucleotidyl transferase (Tdt) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay

Apoptotic cells were determined by TUNEL assay. This assay was performed on testicular tissue sections using ApopTag^® plus peroxidase in situ apoptosis detection kit (Catalogue #S7101, Millipore, Billerica, MA, USA) as per manufacturer’s instruction. Reactions were developed with DAB detection system, and sections were counterstained with Hematoxylin. Stained sections were visualized under light microscope (Nikon Eclipse i80, Tokyo, Japan). Images captured at 200× magnification were used for quantification and are presented in results.

H-Score

The evaluation of the immunohistochemical labeling of Kir6.2, caspase 3 and TUNEL in samples from experimental groups was performed using H-Score. Briefly, sections were evaluated using a light microscope (Nikon Eclipse i80, Tokyo, Japan) with a special ocular scale. Five different fields from all slides at 200× magnification were evaluated for immunohistochemical labeling of Kir6.2, caspase 3 and TUNEL. The labeling was scored in a semi-quantitative fashion that included the intensity of specific labeling in sections. The evaluations were recorded as percentages of labeled cells of all types in each of four intensity categories, denoted as 0 (no labeling), 1+ (weak labeling but detectable above control), 2+ (distinct labeling), and 3+ (intense labeling). For each tissue, an H-Score value was derived by summing the percentages of cells that were labeled at each intensity multiplied by the weighted intensity of the labeling: H-Score = ∑ Pi (i+1), where i is the intensity score and Pi is the corresponding percentage of the cells. Two observers blinded to the experimental groups performed the H-Score evaluations, and the average score was used.

Spermiogram analysis

The cauda part of the left epididymis was cut into small pieces in PBS and the mobilized sperm were collected. The obtained sperm samples were counted with Makler counting chamber under light microscope (Olympus CX21). Sperm motilities were classified as: (i) moving forward; (ii) slow motion; (iii) mobile in place; (iv) immobile.

Biochemical analysis

Blood serums in -80°C were taken to -20°C and waited overnight. Then, samples were taken to +4°C and the temperature was gradually increased. Biochemical analyzes were performed in Biochemistry Research Laboratory, Faculty of Medicine, Bolu Abant Izzet Baysal University. For measuring MDA and testosterone levels, enzyme-linked immunosorbent assay (ELISA) method was used. Reference ranges of 0.1–20 ng/mL for testosterone (Elabscience E-EL-0072) and 31.25–2000 ng/mL for MDA (Elabscience E-EL-0060) were used. Measurements were done on BioRad ELISA reader. The optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm.

Statistical analysis

Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) Statistics 20 program. Groups were compared by using one-way analysis of variance (ANOVA) tests followed by post hoc Tukey’s honestly significant difference (HSD) test. The significance value was accepted as p≤0.05.

⧉ Results

Morphological assessment of testicular tissues

According to our HE-staining, no HP changes were observed in testicular tissues of Sham group (Figure 1a). Similarly, there was no HP defect in testicular tissues of DMSO and Carvacrol groups (Figure 1, b and c). In testicular tissue sections of IR and IR+DMSO groups, there were congestion, hemorrhage, thickening, edema, disruption in seminiferous tubule forms, irregularity in germinal epithelium and cell debris in seminiferous tubule lumen (Figure 1, d and e). Structural defects and congestion in IR+Carvacrol group was less compared to IR and IR+DMSO groups (Figure 1f).

Figure 1.

HE-staining of all groups: (a) Sham; (b) DMSO; (c) Carvacrol; (d) IR; (e) IR+DMSO; (f) IR+Carvacrol. Cell debris is shown with asterisks (*), edema is shown with arrows and hemorrhage is shown with arrowheads. Scale bar: 100 μm. DMSO: Dimethyl Sulfoxide; HE: Hematoxylin–Eosin; IR: Ischemia–reperfusion

According to Masson’s trichrome staining, there were not any HP changes in testicular tissues of Sham, DMSO and Carvacrol groups (Figure 2a,2b,2c). Congestion, thickening, edema, disruption in seminiferous tubule forms, irregularity in germinal epithelium and cell debris in seminiferous tubule lumen were seen clearly in IR group (Figure 2d). Also, hemorrhage was seen in both IR and IR+DMSO groups (Figure 2, d and e). Structural defects and congestion in IR+Carvacrol group was less compared to IR and IR+DMSO groups (Figure 2f).

Figure 2.

Masson’s trichrome staining of all groups: (a) Sham; (b) DMSO; (c) Carvacrol; (d) IR; (e) IR+DMSO; (f) IR+Carvacrol. Cell debris is shown with asterisks (*), hemorrhage is shown with arrows. Scale bar: 100 μm. DMSO: Dimethyl Sulfoxide; HE: Hematoxylin–Eosin; IR: Ischemia–reperfusion

Seminiferous tubule diameter measurements showed that, there was no significant difference between Sham, DMSO and IR groups (p>0.05). Likewise, there was no significant difference between IR, IR+DMSO and IR+Carvacrol groups (p>0.05). The tubule diameter was significantly decreased in IR and IR+DMSO groups compared to Carvacrol group (p<0.05) (Figure 3).

Figure 3.

Mean seminiferous tubule diameters of all groups. Significant difference was accepted as p<0.05. ^aSignificant difference compared to Carvacrol group. DMSO: Dimethyl Sulfoxide; IR: Ischemia–reperfusion

According to data obtained from Johnsen’s scoring, scores were significantly decreased in IR, IR+DMSO and IR+Carvacrol groups compared to Sham, DMSO and Carvacrol groups (p<0.0001). This statistically significant difference showed that IR injury caused seminiferous tubules disruption. There was also statistically significant difference between IR and IR+Carvacrol groups (p<0.001). In IR+Carvacrol group, scores were significantly increased compared to IR and IR+DMSO group’s scores. The increase of Johnsen’s score in IR+Carvacrol group compared to IR and IR+DMSO group may suggest that Carvacrol could prevent damage in seminiferous tubules (Figure 4).

Figure 4.

Mean Johnsen’s scores of all groups. Significant difference was accepted as p<0.05. Significant difference compared to: ^aSham group; ^bDMSO group; ^cCarvacrol group; ^dIR group; ^eIR+DMSO group. DMSO: Dimethyl Sulfoxide; IR: Ischemia–reperfusion

Spermiogram results

According to our spermiogram results there was no significant difference in total sperm concentration between groups (Figure 5a). Our total sperm motility results showed that, there was a statistically significant difference between Sham, DMSO, Carvacrol and IR groups (p<0.0001). Total sperm motility was increased in Carvacrol group compared to Sham group (p<0.05). In IR, IR+DMSO and IR+Carvacrol groups, total number of motile sperm was decreased compared to Sham, DMSO and Carvacrol groups. This data may show that IR injury could cause decrease of motile sperm count. There was no statistically significant difference between IR and IR+Carvacrol groups (p>0.05) also IR+DMSO group (p>0.05) (Figure 5b).

Figure 5.

(a) Mean total sperm concentration of all groups. There was no statistically significant difference between groups, p<0.05; (b) Total sperm motility of all groups. Significant difference was accepted as p<0.05. Significant difference compared to: ^aSham group; ^bDMSO group; ^cCarvacrol group. DMSO: Dimethyl Sulfoxide; IR: Ischemia–reperfusion

Kir6.2 IHC

Kir6.2 immunoexpression was cytoplasmic in all experimental groups. Kir6.2 immunoexpressions were more frequently observed in round spermatids. There was Kir6.2 immunoexpression in primary spermatocytes but intensity was less compared to round spermatids. In Sham, DMSO and Carvacrol groups, Kir6.2 immunoexpressions were low (Figure 6a,6b,6c). Kir6.2 immunoexpression was increased in both IR and IR+DMSO groups compared to Sham, DMSO and Carvacrol groups but it was less than IR+Carvacrol group (Figure 6, d and e). We observed that Kir6.2 immunoexpression was higher in IR+Carvacrol group compared to Sham, DMSO and Carvacrol groups (Figure 6f). H-Score for Kir6.2 IHC is shown in Figure 6g. Kir6.2 immunoexpression was increased in IR, IR+DMSO and IR+Carvacrol groups compared to Sham, DMSO and Carvacrol groups (p<0.0001). This result may show that IR injury can cause the opening of ATP-dependent potassium channels in testis. In IR+DMSO group, Kir6.2 immunoexpression was significantly increased compared to IR group (p<0.05). Kir6.2 immunoexpression was significantly increased in IR+Carvacrol group compared to IR (p<0.0001) and IR+DMSO groups (p<0.001) (Figure 6g). This observation suggests that Carvacrol may have a protective role against IR injury via opening Kir6.2 channels.

Figure 6.

Kir6.2 immunohistochemistry in seminiferous tubules of all groups. Scale bar: 50 μm. (a) Sham; (b) DMSO; (c) Carvacrol; (d) IR; (e) IR+DMSO; (f) IR+Carvacrol; (g) Ki6.2 H-Score. Significant difference was accepted as p<0.05. Significant difference compared to: ^aSham group; ^bDMSO group; ^cCarvacrol group; ^dIR group; ^eIR+DMSO group. DMSO: Dimethyl Sulfoxide; IR: Ischemia–reperfusion

Caspase 3 IHC

Apoptotic cells in testicular tissue were positively immunostained with caspase 3. Caspase 3 exhibited mostly cytoplasmic immunoexpression and a little nuclear immunoexpression in all groups. We observed caspase 3 immunoexpressions especially in cell debris, round spermatids, and primary spermatocytes (Figure 7). In Sham, DMSO and Carvacrol groups, caspase 3 immunoexpressions were low (Figure 7a,7b,7c). Caspase 3 immunoexpressions were increased in IR and IR+DMSO groups compared to Sham, DMSO and Carvacrol groups (Figure 7, d and e). In IR+Carvacrol group, caspase 3 immunoexpression was higher than Sham, DMSO and Carvacrol groups but lower than IR and IR+DMSO groups (Figure 7f). H-Score for caspase 3 IHC is shown in Figure 7g. Caspase 3-positive cells were statistically significantly increased in IR, IR+DMSO and IR+Carvacrol groups compared to Sham, DMSO, and Carvacrol groups (p<0.0001). A statistically significant increase of stained cell number in IR group may show that IR injury could induce apoptosis in seminiferous tubules. In IR+DMSO group, caspase 3 immunoexpression was decreased compared to IR group (p<0.05). Apoptotic cells in IR+Carvacrol group were decreased compared to IR and IR+DMSO groups (p<0.0001) (Figure 7g). These results suggest that Carvacrol could prevent apoptosis during IR injury.

Figure 7.

Caspase 3 immunohistochemistry in seminiferous tubules of all groups. Scale bar: 50 μm. (a) Sham; (b) DMSO; (c) Carvacrol; (d) IR; (e) IR+DMSO; (f) IR+Carvacrol; (g) Caspase 3 H-Score. Significant difference was accepted as p<0.05. Significant difference compared to: ^aSham group; ^bDMSO group; ^cCarvacrol group; ^dIR group; ^eIR+DMSO group. DMSO: Dimethyl Sulfoxide; IR: Ischemia–reperfusion. DMSO: Dimethyl Sulfoxide; IR: Ischemia–reperfusion

TUNEL assay results

TUNEL assay results were compatible with caspase 3 IHC. In Sham, DMSO and Carvacrol groups, TUNEL-positive cells were low compared to IR and IR+DMSO groups (Figure 8a,8b,8c). TUNEL-positive cell numbers were increased in IR and IR+DMSO groups compared to IR+Carvacrol groups (Figure 8d,8e,8f). H-Score for TUNEL assay is shown in Figure 8g. TUNEL-positive cells were decreased significantly in IR+DMSO and IR+Carvacrol groups compared to IR group (p<0.0001). A significant decrease of apoptotic cell number in IR+Carvacrol group may show that Carvacrol could prevent apoptosis during IR injury.

Figure 8.

Determination apoptotic cells with TUNEL assay on seminiferous tubules of all groups. Scale bar: 50 μm. (a) Sham; (b) DMSO; (c) Carvacrol; (d) IR; (e) IR+DMSO; (f) IR+Carvacrol; (g) TUNEL H-Score. Significant difference was accepted as p<0.05. Significant difference compared to: ^aSham group; ^bDMSO group; ^cCarvacrol group; ^dIR group; ^eIR+DMSO group. DMSO: Dimethyl Sulfoxide; IR: Ischemia–reperfusion; TUNEL: Terminal deoxynucleotidyl transferase (Tdt) deoxyuridine triphosphate (dUTP) nick end labeling

Serum MDA and testosterone levels

Serum MDA and testosterone levels were determined by ELISA method in all groups, and the results are shown in Figures 9 and 10. Serum MDA levels were highest in IR group. In Sham, DMSO and Carvacrol groups, MDA levels were lowest. MDA levels also decreased in IR+Carvacrol group. Serum MDA levels were increased in IR (p<0.001) and IR+DMSO (p<0.05) groups compared to Sham, DMSO and Carvacrol groups. These data showed that IR injury caused lipid peroxidation. MDA level was decreased in IR+Carvacrol group compared to IR (p<0.001) and IR+DMSO (p<0.05) groups. The decrease of MDA values in IR+Carvacrol group showed that treatment with Carvacrol reduced lipid peroxidation because of its antioxidant role (Figure 9).

Figure 9.

Mean serum MDA levels in all groups. Significant difference was accepted as p<0.05. Significant difference compared to: ^aSham group; ^bDMSO group; ^cCarvacrol group; ^dIR group; ^eIR+DMSO group. DMSO: Dimethyl Sulfoxide; IR: Ischemia–reperfusion; MDA: Malondialdehyde

Figure 10.

Mean serum testosterone levels in all groups. Significant difference was accepted as p<0.05. ^aSignificant difference compared to Carvacrol group. DMSO: Dimethyl Sulfoxide; IR: Ischemia–reperfusion

Serum testosterone level was highest in Carvacrol group. Also, serum testosterone level was lowest in IR group. Serum testosterone level was significantly decreased in IR group compared to Carvacrol group (p<0.05). In IR+DMSO group, serum testosterone level was decreased compared to Carvacrol group (p<0.05). There was no significant difference between IR, IR+DMSO and IR+Carvacrol groups (p>0.05). The decrease in testosterone levels in all IR groups has shown that IR injury could reduce testosterone level (Figure 10).

⧉ Discussions

Testicular torsion is a urological problem that causes tissue damage and subfertility. When the testis tissue rotates around the spermatic cord and veins, blood flow stops, and tissue damage begins [1]. Testicular torsion–detorsion causes testicular damage because torsion leads to ischemia and detorsion causes reperfusion injury [34]. IR injury leads to increase of ROS, lipid peroxidation and apoptosis in tissues [8]. ROS initiates lipid peroxidation in the cell membrane by attacking polyunsaturated fatty acids [11]. Lipid peroxidation causes testicular dysfunction, sperm anomalies and decreasing capacitation of sperms [12, 16]. ROS, which causes OS, accelerates the release of cytochrome c by disrupting the mitochondrial membranes. With the release of cytochrome c, caspase activation begins, and apoptosis occurs [35,36,37]. KATP channels were first discovered in cardiomyocytes [38]. The opening of these channels is associated with the metabolic status of the cell and the level of ATP [39]. Kir6.2, which is subunit of the KATP, is found in spermatogonium, spermatocyte and late-stage sperm [20]. Opening of KATP protects tissue against IR injury [19]. Carvacrol is a monoterpenoid compound found in essential oils of various aromatic plants [26]. It has antioxidant, antiseptic, antibacterial and antifungal activities [27,28,29]. Carvacrol reduces lipid peroxidation via its antioxidant role [31].

Romeo et al. reported dilatation, edema, and hemorrhage in the intertubular areas of the testicle of IR injury-induced rats [40]. In another study of rat IR injury model, irregularity in the testicular tissue, thinning of the germinal epithelial layers, degeneration of germ cells in the tubules was observed [41]. Aydıner et al. also reported damage in seminiferous tubules and intertubular areas of testicular tissues of rats that IR was induced [42]. Our results are compatible with the literature; we observed that testicular IR injury caused congestion, thickening, edema, impairment of seminiferous tubule structures, abnormalities in germinal epithelium and cell debris in the lumen. All these findings show that IR injury causes HP damage of testicular tissue.

We used Johnsen’s score to evaluate the morphological damage of the seminiferous tubules [33]. When we checked the literature, we found that different research groups found significant decrease in Johnsen’s score of the testicular IR injury group compared to the control group [43,44,45,46]. In our study, there was a significant decrease of Johnsen’s score in IR group compared to Sham group as in other studies. There was a significant increase of Johnsen’s score in IR+Carvacrol group compared to IR group. This result shows that Carvacrol may protect spermatogenic cells and seminiferous tubule structure against IR damage.

Studies showed that sperm motility and sperm concentrations were decreased in IR group rats compared to control group rats [44, 47,48]. In our study, like other studies, there was a significant decrease in the percentage of total sperm motility in IR group compared to control group, but no significant difference was found between groups for sperm concentration. We did not observe any difference in total motile sperm percentage; therefore, we think that if Carvacrol treatment duration is extended, positive effects on sperm motility and concentration could be seen.

Teshima et al. reported that mitochondrial (mito)KATP channels opened with Diazoxide plays a neuroprotective role by preventing apoptosis [49]. Similarly, Tsounapi et al. reported that Kir6.2 was opened after Cromakalim administration and prevented apoptosis in rat testicular IR model [25]. In another study, sarcolemmal (sarc)KATP channel was opened via P-1075 and it reduced lipopolysaccharide-induced apoptosis, while blocking sarcKATP channel with HMR-1098 increased lipopolysaccharide-induced apoptosis in cultured neonatal rat cardiomyocyte cells [50]. Oliveira et al. described that Carvacrol mediates the release of endogenous prostaglandins on an experimental gastric lesion model in rodent, which has a gastroprotective role by causing increased mucus production, opening of KATP channels, activation of nitric oxide (NO) synthase [51]. Our results support the findings of Tsounapi et al. and Oliveira et al. [25, 51]. It is presumed that Kir6.2 KATP channels are opened in IR injury and Carvacrol administration could help prevent IR injury by further activating opening of these channels.

Different research groups reported the increase of TUNEL-positive cells and caspase 3 immunoexpressions in testicular IR injury-induced rats compared to control and Sham group rats [52,53]. Similarly, in traumatic neuronal injury in vitro model, Carvacrol treatment significantly decreased TUNEL-positive cell number and caspase 3 immunoexpressions compared to injury group [54]. Suo et al. reported that Carvacrol treatment decreased TUNEL-positive cell number and caspase 3 activation in liver IR injury model induced rats [55]. In another study, Carvacrol treatment significantly reduced the number of TUNEL-positive cells in kidney tissues of mice that were treated with Cisplatin [56]. We obtained similar results in our study as well. We observed caspase 3 immunoexpressions especially in cell debris in the lumen, round spermatids, and primary spermatocytes. Apoptotic cells had a nuclear staining with TUNEL assay. Our results may show that testicular IR injury initiates apoptosis, while Carvacrol prevents apoptosis during IR injury.

The increase of MDA levels as a marker of lipid peroxidation in testicular IR injury-induced rats compared to control group rats was shown in different studies [57,58,59]. Yu et al. indicated significant increase of MDA levels in acute myocardial infarction model induced rats compared to Sham group rats, while Carvacrol administration decreased MDA levels [60]. Samarghandian et al. reported significant increase of MDA levels in age-related OS damaged livers of rats and significant decrease of MDA levels in Carvacrol treatment group [61]. In liver of IR, injury model induced rats MDA levels were significantly increased compared to control group, and MDA levels were significantly decreased in Carvacrol-treated group [55]. Cetik et al. reported a significant decrease in MDA levels after Carvacrol administration in their cardiotoxicity model [62]. In our study, our findings were similar to previous studies; we found a significant increase in MDA levels in IR group. We also observed a significant decrease of MDA levels in Carvacrol administrated IR group. We can comment that testicular IR injury causes lipid peroxidation and Carvacrol treatment may protect against IR damage and reduce lipid peroxidation.

In the literature, effects of DMSO on MDA levels were shown. Sahin et al. reported that DMSO caused to decrease MDA levels in liver of IR injury-induced rabbits compared to untreated IR group rabbits [63]. Tamjidipoor et al. showed MDA levels were increased after ovariectomy and decreased after DMSO application in rats [64]. Li et al. indicated that Zymosan injection increased MDA levels and DMSO suppressed the increase of MDA in Zymosan-induced intestinal inflammation model in rats [65]. We observed that DMSO, which we used as a solvent, decreased MDA levels. All these studies may show that Carvacrol and DMSO prevent lipid peroxidation.

There are studies showing decrease of serum testosterone levels after IR in testicular tissue [66,67]. Some studies also show increase of testosterone levels after different treatment agents. Gholami et al. reported a significant decrease of testosterone levels in IR group and a significant increase in honey administrated testicular IR model group, 50 days after reperfusion [68]. Araghi et al. reported increase of serum testosterone levels after Carvacrol administration in mouse with Ketamine-induced testicular damage [69]. Other studies and our study may show that IR injury reduces testosterone levels. Despite two hours of ischemia and two hours of reperfusion and Carvacrol administration, serum testosterone levels did not change. To see whether Carvacrol influences testosterone levels, hormone levels can be measured after long-term Carvacrol treatment.

⧉ Conclusions

Taken together, IR injury causes HP damage of testicular tissue and spermatogonial cells. There was a significant decrease in total motile sperm percentage in IR group. There was no statistically significant difference for total motile sperm percentage between IR and IR+Carvacrol group, but Carvacrol treatment led to an increase of approximately 3% in the rate of motile sperm in IR+Carvacrol group, which has an important value in terms of assisted reproduction techniques. There was no significant difference for sperm concentration between groups. To better understand the effect of Carvacrol on sperm concentration and motility, Carvacrol administration time should be extended. A significant increase of Kir6.2 activation was detected in IR group. In IR+Carvacrol group, Kir6.2 activation was significantly increased compared to IR group. As a result, it is concluded that Carvacrol has a KATP channel opening effect. Caspase 3 immunoexpression was increased in testicular IR injury-induced group, and it was decreased in Carvacrol-treated group. This suggests that Carvacrol may have an anti-apoptotic effect. There was a significant increase in TUNEL-positive cell number in IR group, and a significant decrease in IR+Carvacrol group. Carvacrol may have protective properties against germ cell death. In testicular IR injury model, it has been observed that Carvacrol has an antioxidant role and significantly reduces MDA levels and prevents OS. A minimal increase in serum testosterone values was observed in the IR+Carvacrol group as a treatment, but no statistically significant difference was found. To understand the effect of Carvacrol on testosterone level, the hormone level should be measured in the tissue or at the end of long-term treatment and administration. Studies have shown that Carvacrol has various bioactivities, such as antiviral, antifungal, antioxidant. Our study showed that Carvacrol, as an antioxidant, reduces lipid peroxidation by inhibiting ROS in testicular IR damage, inhibits apoptosis and acts as a KATP channel opener, thus reducing ischemic damage.

Conflict of interest

The authors declare that they have no conflict of interests.